Evaluation of Responses Embryogenic Cycas Revoluta Thumb., from Callus Culture Obtained in Vitro

Journal of Applied Biotechnology & Bioengineering

Research Article Open Access Evaluation of responses embryogenic Cycas revoluta thumb., from callus culture obtained in vitro

Summary Volume 6 Issue 4 - 2019 Processes cell differentiation and dedifferentiation are included in the development of 1 2 biotechnology protocols to promote somatic embryogenesis as an alternative to the in vitro Rodríguez de la O JL, Lazarous J Flores VA, propagation of plants, somatic embryos may be an excellent strategy for both propagation Robledo Monterrubio MS,3 Contreras Cruz F3 and conservation of fossil species such as cycads. They were evaluated in vitro with 1Department of Plant Science, Autonomous University Chapingo, different strategies, morphogenic responses associated with obtaining somatic embryos Mexico 2 of C. revolute. calli of megagametophytes, subsequently subcultured in four combinations Thesis Master of Science in Biotechnology, Department of Plant of basic salts of Murashige and Skoog (1962) MS, with the addition of benzyladenine Science, Mexico 3 (BA), and 2, dichlorophenoxyacetic (2,4-D) were used, and kinetin (K), and picloram. Professors Autonomous University Chapingo, Mexico In the results, It was possible to characterize potentially embryogenic callus, evaluating Correspondence: Rodríguez de la O JL, Academy of genetics, the levels of both cellular differentiation, necrosis, texture and color; and increases mass Department of Plant Science, Autonomous University Chapingo, or weight considered to start differentiation or proembryogenic or globular type. Calli MexicoCarr. Fed. Mexico Texcoco Km 38.5 CP 56230, Mexico, were subcultured in a medium containing MS salts, incorporating abscisic acid (ABA) in Email 0, 0.38, 1.13, 3.78 and 5.67 uM doses influenced both the production and maturation of somatic embryos. Embryonic structures, presented a pink coloration characteristic strongly Received: July 10, 2019 | Published: August 12, 2019 associated towards maturity. The effect of combinations of BA, Kin, 2,4-D, GA3 and ANA influenced the development and germination of mature somatic embryos. And the combination of 1.36 mM 2,4-D+4.44 uM BA promoted the appearance of calluses with a compact texture, characteristic related to their embryogenic potential. The purpose of this research in Cycas sp was to contribute to the study of the in vitro morphogenic responses of this group of plants. And somatic embryogenesis, will allow the obtaining and multiplication as well as its preservation of Cycas sp. Gender that is evolutionary very important.

Keywords: C. revoluta, cycad, somatic embryogenesis, growth regulators

Introduction years and that the use of haploid explants or those that do not require fertilization, can significantly optimize genetic resources, since there Cycads are a group of ancestral dioecious, which appeared in are species such as Encephalartos woodii Sander., of which only the pérmico and evolved from the progymnosperms free spores, exemplary and have male sexual propagation is impossible. C. revolute preceding the Gingkoales and Gnetales, succeeded in developing investigations have sought regeneration from ZE;8,9 leaf midrib and mechanisms of adaptation and survival, so have been called “living epicotyls;10 endosperm11 and megagametophytes; however, the use fossils”. Biologically are very interesting because they represent of megagametophytes as a source of explant organogenics responses an important stage in the evolution of flowering plants and are generated (buds and roots), according Ashmore12 are limited for 1 considered baseline in the evolutionary tree seed plants. Their beauty conservation of genetic resources use due to genetic instability and rarity, has generated a great demand, so we have become perfect inherent to these plants. EPE development from embryogenic cultures target of ends and people dedicated to the black market collectors of in cycads, It remains inefficient since cultures cannot be synchronized exotic species, causing today all species of the order Cycadales are and therefore certain developmental events are difficult to identify. included in Appendix I or II of the Convention on International Trade Necrosing problems are frequent and slow responses due to the nature in Endangered Species of Wild Fauna and Flora (CITES, 2017). C. of this type of plant; Litz et al.,7 have indicated that several species of revolute It is the oldest living cycads and is included in Appendix II embryogenic cultures have grown cycad vigorously and are highly of CITES (2017). The species symbolizing economic importance of morphogenic after 11 years of induction. Growth regulators can 2 order, ornamental and besides, they have been attributed to nutritional promote morphogenesis even if the salt concentration is not adequate. and medicinal properties with clear antibacterial and deleterious However, when it is desired to develop ES, use of regulators depends 3–5 effects against colon cancer and epidermoid. However, due to slow on the stage of the process (induction, proliferation, maturation growth, dioicismo, development, and contradictions in the seed, the or germination). While in cycad it has suggested the use of 2,4-D spread is directed to the use of buds or buds in the basal part of the induction, Embryogenic cultures should be subcultured in the same parent plants limiting genetic variability significantly. Thus, It has formulation of basal medium but lacking growth regulators. Litz et emerged a clear need and unpostponable the use of biotechnological al.,7 they founded that these embryogenic cultures are not receptive tools that enable effective propagation of the species to meet demand, to ABA because of its evolutionary condition in the plant kingdom. and contribute to the conservation and avoid extinction. The ES is However, it is essential to remember that many factors other than the in vitro development of embryos from cell culture are not the growth regulators, influencing embryogenic responses, such as the 6 product of a gametic fusion. In the last 70 years are few reports in species and type of explant. Therefore, the objective of this study vitro cultivation in these plants. Somatic embryogenesis in cycads is was to establish the conditions in vitro that can promote the main 7 relatively low and the best responses were obtained from ZE. The morphogenic responses associated with obtaining somatic embryos of morphogenic potential tissue in cycads has been recognized for many

Submit Manuscript | http://medcraveonline.com J Appl Biotechnol Bioeng. 2019;6(4):188‒197. 188 © 2019 la et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and build upon your work non-commercially. Copyright: Evaluation of responses embryogenic Cycas revoluta thumb., from callus culture obtained in vitro ©2019 la et al. 189

Cycas revolute Thunb., Which allow to establish a protocol alternative obtaining globular structures or proembriogenics. Embryogenic calli propagation and preservation and contribute to study of these species. were subcultured to promote both their differentiation and embryonic responses using different strategies based, the medium with the Materials and methods inorganic salts of Murashige & Skoog14 MS (1962), by changing the The research was conducted at the Laboratory of Plant Tissue presence or absence of auxiliaries and regulators increase. Culture Department of Plant Science at the Autonomous University Chapingo, Mexico. calli three months old were used obtained from a. Strategy 1: MS salts (1962) with the source of nitrate in vitro culture of C. revolute megagametophytes from a culture diluted to 50%, supplemented with myo-inositol, thiamine, medium with macroelements and salts of B5 medium13 and microcells pyridoxine, folic acid, biotin, mannitol, L-cysteine, PVP MS medium (1962) supplemented with arginine, asparagine and (100, 0.40, 0.5, 0.5, 0.5, 170 , 60 and 70 mg L-1, respectively) glutamine (100, 100 and 400mg L-1, respectively), 6% sucrose and and 4% sucrose. 6 g L-1 gellam-gum. Calli were obtained under the effect of two b. Strategies 2: MS (1962) 100% salts, supplemented with combinations of auxin-cytokine (Table 1). The process for promoting thiamine, myo-inositol, glutamine, arginine, asparagine somatic embryogenesis was evaluated in several experimental stages (0.40, 100, 400, 100 and 100 mg L-1, respectively) and 6% and each strategies., Considered as the induction, maturation and sucrose. germination of embryos obtained. c. Strategy 3: MS (1962) diluted to 50% salts, supplemented with myo-inositol, thiamine, pyridoxine, folic acid, biotin, Table 1 Types and auxin-cytokine concentrations applied to megagametophytes mannitol, PVP (100, 0.40,0.50, 0.50, 0.50, 170 and 70mg of Cycas revolute for callus induction L-1) and sucrose 4%. d. Strategy 4: MS (1962) salts, reducing the source of nitrate Treatment Growth regulators (uM) 50%, supplemented with myo-inositol, thiamine, pyridoxine, 2,4-D picloram Kin BA TDZ folic acid, biotin, mannitol, PVP (100, 0.40,0.50, 0.50, 0.50, 170 and 70mg L -1) and 4% sucrose. one 9.05 - 13.93 - - e. The media were adjusted to pH 5.7±0.1, the effect of three two - 27.75 - 2.13 2.22 different combinations of growth regulators and a control (Table 2) was tested. Promotion stage callus. Mass increases, cell differentiation, and Table 2 Types and concentrations of growth regulators applied to amorphous callus Cycas revolute in proliferation step

Growth regulators employees (uM) Strategies Treatment BA 2,4-D Kin picloram 0 - - - - one 1.33 0.45 - - one two 4.44 1.36 - - 3 13.32 4.52 - - 0 - - - - one - 0.45 2.32 - two two - 2.26 4.65 - 3 - 6.79 13.94 - 0 - - - - one - - 0.46 4.14 3 and 4 two - - 2.32 4.14 3 4.65 4.14

Variables evaluated To identify the physical characteristics of callus associated with their embryogenic potential was established a model to determine levels of cellular organization or differentiation, until the formation of globular structures or proembriogenics type, having as variables. 1. Levels cell differentiation (DC), with: 0, no differentiation (callus); 1, minimal differentiation; 2, cell aggregates 3. Consistency: friable or compact. (globular or pro embryonal appearance); and 3, the presence of globular or embryonic structures (EPE). 4. Levels of blackening necrosing (oxidation) (NO), with: 0, 25% less necrosing or oxidation; 1, 25 to 75%; and 3, greater 2. Coloring callus, based on the scale of Pantone. The colors than 75% of the developed callus. used were: 5. And the number of distinct globular structures or proembrionarias (EC).

Citation: la RDOJL, Flores LJVA, Monterrubio RMS, et al. Evaluation of responses embryogenic Cycas revoluta thumb., from callus culture obtained in vitro. J Appl Biotechnol Bioeng. 2019;6(4):188‒197. DOI: 10.15406/JABB.2019.06.00192 Copyright: Evaluation of responses embryogenic Cycas revoluta thumb., from callus culture obtained in vitro ©2019 laet al. 190

Increase in mass (weight) of the callus (IB) completely randomized design with 10 replications per treatment. Each experimental unit was a Gerber type flask with 20 ml of culture Both the number of embryonic structures and levels of cell medium. differentiation DC, and the levels of blackening or oxidation and color callus were evaluated every ten days using a stereomicroscope Carl Variables evaluated Zeiss Stemi DV4 with a lens (8x/21) for 80dds. IB biomass increases As variable was the formation of mature somatic embryos obtained in corns, was taking his weight using an analytical balance every every ten days, counted with a stereomicroscope Stemi DV4 Carl month for three months. In each experimental distribution strategy Zeiss. Calluses They were incubated with 16 hour photoperiod light remain four treatments a Table 2 and with 10 repetitions per treatment. Each experimental unit was a Gerber type flask with 20 ml of culture (26°C) and 8 hours darkness (24°C). medium. Stage, germination and development of somatic Obtaining step and embryo maturation embryos Mature somatic embryos were subcultured on MS basal medium Callus cell differentiation levels DC were transferred to 3/2 MS (1962) with a pH of 5.7±0.1, supplemented with myo-inositol, medium (1962) by diluting 75 and 25% of nitrate source (N03) and thiamine, pyridoxine, folic acid, biotin, Polivinylpilorridone (PVP), ammonium (NH4), respectively. The medium with a pH of 5.7±0.1 mannitol (100, 0.40, 0.50, 0.50, 0.50mg L-1), 4% sucrose and 7mg L-1 was added glycerol (30 g L-1), glutamine, arginine, asparagine, agar. Evaluating the percentage of embryos with responses associated L-cysteine, PVP (400, 100, 100, 60 and 70mg L-1) and sucrose with maturity and germination, evaluating the set of auxins, cytokinins one%. Incorporating Ac. Abscisic (ABA), in concentrations of and gibberellins (Table 3) effect. Calli were incubated under the same 0, 0.38, 1.13, 3.78 and 5.67μM. The experiment was set under a conditions of light and temperature in step above.

Table 3Types and concentrations of growth regulators used in the germination of somatic embryos of Cycas revolute

Growth regulators Treatment (μM) 2,4-D BA Kin ANA AG3 0 - - - - - one - - 4.65 1.61 1.44 two - - 13.93 5.37 2.88 3 1.33 0.45 - - - 4 4.44 1.35 - - - 5 13.32 4.53 - - -

Statistic analysis can promote embryogenic responses, even when the concentration of salts is not adequate. To determine the effect of growth regulators in vitro responses embryogenic callus cultivated on an analysis of variance for variables Quantification of globular structures or CE and IB, the proliferation phase is performed; and number of proembrionarias and (IB) mature embryos with a completely randomized design with Tukey test with α=0.05 and comparison of means using SAS (Statistical Analysis Strategy 1: Nitrate reduction 50% MS medium (1962), did not System, 2002). influence the formation of structures proembrionarias EPE, before 80 dap; however, the combination of regulators BA+2,4-D promoted The statistical model used was: enhanced differentiation of embryonic structures. Noting that the Yij =++µ Tij eij top level of cytokinin BA, limits the growth of calluses in their increasing mass, but promotes cell differentiation processes, to obtain where: Yij, Variable Response; μ, average general; Tij, Effect of proembrionarias structures (IB), as shown by the results with α=0.05 growth regulators; ij, experimental error. , that there were significant differences in the number of EPE arising For variables: DC levels, blackening, necrosing (oxidation), color under different doses employed BA+2,4-D (Table 4); However, the and consistency, the proliferation stage, pictures with the Chi-squared variability obtained is due to the effect of treatments, as described Pearson (X2) were performed. Fieire (2003), which explains that embryonic responses are grouped characteristics callus cultured.he combination of 4.44 uM +1.36 Results and discussion (BA+2,4-D), had a higher response in obtaining embryonic structures (average 2.30), at 80dds. (Figure 1). Thus, the behavior was callus The calli were subcultured in basic medium with the inorganic EPE concerning the number of the most obvious development period. salts of MS (1962), for each of the steps aimed at obtaining somatic embryos, and according to the results obtained, the concentration Strategy 2: The basic MS medium (1962) over the combination of of salts and mainly the type and concentration growth regulators kinetin+2,4-D promoted more EPE compared to other strategies co employed influenced decisively on this particularly that regulators callus culture in the differentiation stage. Statistically significant

differences (α=0.05) IB when the concentrations of kin+2,4-D level of DC (2) having 50%; nodular characteristics and little response increased; however, compared to the results obtained in strategy 1, to the formation EPE. However, statistically significant differences treatment stimulated greater IB, also generated a higher number of with α=0.05 between the necrosing and the texture type and coloring EPE (Table 5); recognizing the effect of promoting embryogenesis according to the concentrations of growth regulators used, including due to the presence of 2,4-D coinciding with Cangahuala et al.,15 the control (Table 8) were presented. Accepting cytokinins high doses and Konieczny et al.,16 that evaluated this effect, regarded as one of promotes accumulation of phenolic compounds by Abohatem. So the factors that will determine the ES promotion rates. Comparison 13.32μM BA presented 3 necrosing levels. The effect of kin+2,4-D of means showed greater variability treatment, indicating precisely in the MS medium (1962), promoted the development of calluses a one ESAF. The number of EPE was a significant increase according DC level 2; However, unlike strategy 1, it expressed a clear friable to the concentration of growth regulators used, evident from dds 40 texture in 72.5% of the calli and necrosing of 47.5%. I exist significant (Figure 2). differences with α=0.05 in the color corns between treatments, including the control (Table 8). MS basal medium (1962) with reducing Table 4 Effect of four levels BA +2,4-D on the amount of callus and number salts 50%, and the effect of kin+picloram, calli promoted structures of structures proembriogenics Cycas revolute to 80 dap with differing levels of 45% and compact texture at 70%; however, Concentration Number of different Biomass increase (mg) statistically significant differences, with α=0.05 in the necrosing BA + 2,4-D (M) structures between treatments, including the control (Table 8). Incorporating 0 + 0 88.44 too▪ 0.00 b▪ joint kin+picloram to MS medium (1962), 50% reducing nitrate 1.33 + 0.45 86.82 to 0.00 b source, compact calli produced by 72.5%. Statistically significantly different at α=0.05 level of DC (Figure 5). 4.44 + 1.36 72.26 to 2.30 to Table 6 Effect of four levels kin + picloram (50% salts) on the amount of callus 13.32 + 4.52 61.79 to 0.8 ab and average number of proembrionarias structures Cycas revolute to 80 dap ▪Valores with the same letter within columns are equal according to the Tukey Number of test at P≤0.05. Concentration of Biomass increase (mg) different kin + picloram (uM) Table 5 Effect of four levels kin + 2,4-D on the amount of callus and number structures of structures proembrionarias Cycas revolute to 80 dap 0 + 0 109.9 a▪ 1.30 too▪ Number of Concentration Biomass increase (mg) different 0.46 + 4.14 580.9 to 1.70 to KIN + 2,4-D (μM) structures 0 + 0 44.00 b▪ 0.00 too▪ 2.32 + 4.14 390.8 to 5.50 to

2.32 + 0.45 73.74 b 2.10 4.65 + 4.14 321.6 to 3.50 to

4.65 + 2.26 ab 143.53 2.70 to ▪Valores with the same letter within columns are igualdes according to the 13.94 + 6.79 236.96 to 10.00 Tukey test with a P≤0.05. Table 7 Effect of kin + picloram levels (source of nitrate 50%) on the amount ▪Values with the same letter in column are equal according to the Tukey test and number of callus proembrionarias structures Cycas revolute to 80 dap at P≤0.05. Biomass Concentration of Proembrionarias Strategy 3: MS (1962) medium diluted 50%, the average increase kin + picloram (uM) number of structures development allowed EPE 1.30 to 80 dds in the absence of growth (mg) regulators. Meanwhile combining 2.32+4.14 uM (kin+picloram) 0 + 0 136.2 b▪ 1.10 b stimulated average 5.5 differentiated structures (of which 25.45% were poorly differentiated structures development. The increased cell 0.46 + 4.14 803.8 ab 5.50 to differentiation under the effect of 0.46+4.14 mM (kin+picloram), was 2.32 + 4.14 925.6 to ab 2.80 reflected in a numerically greater IB, the high concentration auxin versus cytokinin source (Table 6). Production structures differentiated 4.65 + 4.14 659.3 ab 1.90 b under the effect of 2.32+4.14 uM (+picloram kin) was higher after 40 dds (Figure 3). ▪Values with the same letter in column are equal according to the Tukey test with a P≤0.05. Strategy 4: The basic MS medium (1962) diluting nitrate 50% without the presence of growth regulators, allowed development of Obtaining somatic embryo maturation average 1.10 EPE. Moreover employed combinations of kin+picloram The combination 4.44μM 1.36μM BA+2,4-D and diluting developed statistically significant responses (α=0.05) in IB and MS inorganic salts (1962) 50%, potentially promoted obtaining number of EPE dds 80 (Table 7). Figure 4 shows the production of embryogenic callus, to 170 dds, with differing levels of DC (2), color EPE upward under the effect of the doses used kin+picloram from (2) and compact texture (Figure 6), initially calluses were cultured 40 dds. on MS medium (1962), without the presence of growth regulators, Physical characteristics of calluses and light. The presence of regulators in the doses mentioned, promoted embryonic structures from 10 dds and pink. Differentiation The combination of BA+2,4-D in the MS (1962) with the source was progressive from 40 days (Table 9); and maturity from 80 dds. of nitrate diluted to 50%, stimulated the development of callus with a

According to their histology was observed on meristematic activity responses were not satisfactory due to the presence of auxin in some of cells in apical zones. 6, the development of pre-cotyledon and treatments inhibiting subsequent embryonic development as reported cotyledon structures presented in some somatic embryos (Figure Vinas and Jimenez.16 Also, tripe with DC (2) level less than 100 days 8). Embryogenic developments C. hildae obtained from the culture old, were subcultured in the same medium and showed no embryonic of leaves of young leaves.EPE 80, 60, and 100 dds, generated in the responses, indicating that the time of the callus culture is essential in means of proliferation stage were subcultured to maturity; however, obtaining somatic embryos, as reported by Ruiz.18 Table 8 Significance obtained by Pearson X2 test in the evaluation of statistically significant contrast between treatments mode in proliferative stage callus Cycas revolute to 80 dap

variables

statisticians Levels of cell Necrosing MS mode (1962) Coloration Consistency differentiation levels

one P-value 0.13 0.01* 0.00* 0.01* X2 2.51 2.26 0.02 0.09 two P-value 0.71 0.17 0.01* 1.00 X2 10.80 2.86 0.84 - 3 P-value 0.18 0.03* 0.20 0.95 X2 2.90 1.25 5.38 8.00

4 P-value 0.03* 0.04* 0.01* 0.48

X2 1.27 3.13 4.81 2.27

*, P≤0.05 statistically significant differences between treatments. The use of ABA in culture medium showed that the higher the dose, plant kingdom. The observation of the gradual development of the lower the number of developed somatic embryos (Table 9); somatic embryos obtained from C. revolute, allowed to show their meanwhile, von Arnold et al.,19 mentioned that ABA can promote morphological development is similar to that expresses a ZE, since the synthesis of storage reserves in embryos during maturation; the development of pro-embryos (being those which showed internal however, Litz et al.,7 reported that somatic embryos in cycads are progress) erythrocyte and Torpedo type until the pre and cotyledon not receptive to ABA because of its evolutionary condition in the stage (Figure 7). Table 9 Maturation of somatic embryos of Cycas revolute expressed in the maturation medium under five concentrations of ABA

Days after subculture (dds) ABA dose (μM) 10 twenty 30 40 0 1.70 2.60 3.00 3.00 0.38 1.00 1.30 1.90 2.20 1.13 1.00 1.20 1.70 1.90 3.78 0.60 0.90 1.20 1.40 5.67 0.60 0.80 0.90 0.90

Figure 1 Schematic comparison of the development of EPE (40-60 dds) of Cycas revolute, under the effect of BA +2,4-D.

Figure 2 Schematic comparison of the development of EPE (40-60 dds) of Cycas revolute, under the effect of kin+2,4-D.

Figure 3 Schematic comparison of the development of EPE (40-60 dds) of Cycas revolute, under the effect of kin+picloram (inorganic salts 50%).

Figure 4 Schematic comparison of the development of EPE (40-60) dds Cycas revoluta, under the effect of kin+picloram (source of nitrate 50%).

Figure 5 Development of Cycas revolute proembrionarias structures 70 days old at the MS (1962) proliferation step. A) 4.44+1.36 uM (BA+2,4-D; nitrate 50%); B) 13.94+6.79 uM (kin+2,4-D, salts 100%); C) 2.32+4.14 uM (kin+picloram, salts 50%); D) 2.32+4.14 uM (kin+picloram; nitrate 50%).

Figure 6 A) Embryogenic calli of Cycas revolute obtained by combining 4.44 uM BA+1.36 .mu.M 2,4-D; B) Polyembryony observed in medium without growth regulators.

Figure 7 Embryogenic callus responses in Cycas revolute in medium without growth regulators; A) development of rosacea coloration and pro-embryos (10 dds); B) globular embryonic structures immature (30 DAP); C) Callus (70 dds); D) differentiation of somatic embryos with (70 dds) suspensor evident; E) the presence of pre-cotyledon structures (70 dds); F) cotyledon (110 dds). Germination of somatic embryos conversion process plant (germination). Development related to leaf development and the apparent photosynthetic activity could be Embryonic structures 129 days old, had responses associated observed (Figure 8) structures, which coincides with Litz et al.,7 who with the development and germination by 90% under the combined reported that the expansion of the first sheet always circinada or after effect of 4.44+1.35 uM (BA+2,4-D). Germination is a naturally slow germination. process; and also under in vitro conditions; in Figure 8, embryonal observed at 10, 40 and 220 dds, whereas short term are favorable The production of embryonic structures derived from apical compared to ZE responses, which develop from 1 to 1.5 years under meristem area growth mature somatic embryos from 30 dds was in situ conditions. The percentage of germination of somatic embryos evident (Figure 10), averaging 8.7 and 12.1 somatic embryos under combining kin, ANA, AG3, BA and 2,4-D at 40 and 210 dds, shown the effect of 1.33+0.45 and 4.44+1.35 (μM) BA+2,4-D, respectively. in Figure 9, where obviously the medium without the presence of This, evidenced a recurring somatic embryogenesis (ESR), coinciding growth regulators had no effect positive in embryonic development; with who point out that mature somatic embryos are allowed to form comparing the combination 4.44+1.35uM (BA+2,4-D). new embryos derived from epidermal cells. However, the 2.48% exhibit deformations in morphology (fan shape), and promptly let Embryonic development and responses associated germination this kind of derangement may be related to bipolar endogenous auxin were observed in embryos meristematic cell division in apical region transport by Hiraga et al.20 type. Which shows an embryonic indispensable maturation in the

Figure 8 Production of somatic embryos of Cycas revolute with the effect of 4.44+1.35 uM (BA+2,4-D). ab) bipolar meristematic growth evident from the 10 (A) and 40 dds (B); apical meristem growth and radical 10 (C) and 220 dds (D); E) coleoptilar expression of an embryo of 27 dap; F) development of primary leaflets, apparent photosynthetic activity at 70 dds (F).

Figure 9 Combination regulators and blackening levels of cultured in vitro responses observed according to their viability tissues to promote somatic embryogene.

Figure 10 Percentage of responses associated with germination of somatic embryos of Cycas revolute at 40 and 210 dds.

Figure 11 AB) blackening (oxidation) of the root tip apparent somatic embryos of Cycas revolute to 220 dds; CD) embryonic globular structures generated from ESR. Conclusions 2014;10(2):83–92. The combination of 4.44+1.35 uM BA+2,4-D in MS basal 7. Litz RE, Moon PA, Benson MS, et al. A Biotechnology Strategy for Medium- and Long-Term Conservation of Cycads. The Botanical Review. medium (1962) with 50% reduction in the source of nitrate, 2004;70(1):39–46. stimulate the formation of potentially embryogenic callus at 170 days of cultivation, which characterized by a creamy color, compact 8. Motohashi T, Toda M, Kondo K. Adventitious embryo formation from consistency and nodular appearance.MS basal medium (1962) zygotic embryos derived in Cycas revolute. Plant Biotechnology. with NH4NO3 and KNO3 reduction at 75 and 25%, respectively 2008;25(6):589–591. stimulated the production of somatic embryos from 30 days; 9. Pan S, Neeraj A, Srivastava KS, et al. 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